Regulations of important biological functions, such as homeostasis, reproduction, individual development, metabolism, growth, regulations of nervous system, cardiovascular system, immunological system, digestive system and metabolic system, and sensory system are conducted through corresponding responses of cells which accept various endogenous factors such as hormones and neurotransmitter, or organoleptic stimulants such as light and odor via receptors specific thereto on cell membranes. Many receptors of hormones and neurotransmitters involved in these regulations of functions are coupled with guanine nucleotide-binding proteins (hereinafter sometimes referred to as G-proteins) and activate the G-proteins to transmit signals into cells, which then express a variety of functions. In addition, these receptor proteins possess commonly seven transmembrane domains and are thus generically referred to as G-protein-coupled receptors or seven-transmembrane-type receptors. It is known that the interaction of various existing hormones and neurotransmitters with receptor proteins thereof plays a significant role in such regulations of biological functions. However, there are still many unclear points as to the existence of unidentified active substances (e.g. hormones, neurotransmitters) and receptors thereof.
In recent years, sequence information have been accumulated by determination of human genome DNAs or cDNAs derived from various human tissues, and genetically analytical techniques have also been advanced, and as a result, human genes have been elucidated at an accelerating pace. Correspondingly, there are many genes detected, which are predicted to encode proteins with their functions still unknown. G-protein-coupled receptors can be clearly selected from these proteins on the basis of not only presence of seven transmembrane domains, but also presence of many common nucleic acid sequences or amino acid sequences. On the other hand, such G-protein-coupled receptors have also been obtained by Polymerase Chain Reaction (PCR) using the structural similarity as described above. Among G-protein-coupled receptors thus obtained until now, some are a subtype receptor having high structural similarity to known receptors, and thus their ligands may be easily predicted. In most cases, however, it is impossible to predict their endogenous ligands, and the ligands of these receptors have not been found yet. In this connection, these receptors are referred to as orphan receptors. The unidentified endogenous ligands of these orphan receptors may be involved in biological phenomena, which are not well characterized due to the unidentification of the ligands. Thus, if such ligands are associated with important physiological actions and pathological conditions, it is expected that development of agonists or antagonists of the receptors may lead to creation of innovative pharmaceuticals (Stadel, J. et al., TiPS 18, 430-437, 1997; Marchese, A. et al., TiPS 20, 370-375, 1999; Civelli, O. et al., Brain Res. 848, 63-65, 1999). But, not so many ligands of orphan G protein-coupled receptors are actually identified until now.
Recently, some research groups made an attempt to search for ligands of these orphan receptors, and reported isolation of new physiologically active peptides and/or determination of their structures. Reinsheid et al. and Meunier et al. separately isolated a novel peptide called orphanin FQ or nociceptin from pig brain extract or rat brain extract using as an index the response in animal cells expressing an orphan G-protein-coupled receptor LC132 or ORL1 by induction of cDNA encoding either into the cells, and determined the sequence thereof (Reinsheid, R. K. et al., Science 270, 792-794, 1995; Meunier, J. C. et al., Nature 377, 532-535, 1995). It was reported that this peptide was involved in pain sensation, and further shown that the peptide was involved in memory as the result of investigation of the receptor-knockout mice.
Then, until now, novel peptides such as PrRP (prolactin releasing peptide), orexin, apelin, ghrelin, and GALP (galanin-like peptide) have been isolated as ligands of orphan G-protein-coupled receptors by the same methods as described above (Hinuma, S. et al., Nature 393, 272-276, 1998; Sakurai, T. et al., Cell 92, 573-585, 1998; Tatemoto, K. et al., Bichem. Biophys. Res. Commun. 251, 471-476, 1998; Kojima, M. et al., Nature 402, 656-660, 1999; Ohtaki, T. et al., J. Biol. Chem. 274, 37041-37045, 1999).
On the other hand, some of receptors of physiologically active peptides, which had not been clarified before, were identified in a similar way. GPR38 was found as a receptor of motilin which was involved in intestinal contraction (Feighner, S. D. et al., Science 284, 2184-2188, 1999). In addition, SLC-1 was identified as a receptor of Melanin Concentrating Hormone (MCH) (Chambers, J. et al., Nature 400, 261-265, 1999; Saito, Y. et al., Nature 400, 265-269, 1999; Shimomura, Y. et al., Biochem. Biophys. Res. Commun. 261, 622-626, 1999; Lembo, P. M. C. et al., Nature Cell Biol. 1, 267-271, 1999; Bachner, D. et al., FEBS Lett. 457, 522-524, 1999), and GPR14 (SENR) was reported as a receptor of urotensin II (Ames, R. S. et al., Nature 401, 282-286, 1999; Mori, M. et al., Biochem. Biophys. Res. Commun. 265, 123-129, 1999; Nothacker, H.-P. et al., Nature Cell Biol. 1, 383-385, 1999; Liu, Q. et al., Biochem. Biophys. Res. Commun. 266, 174-178, 1999). MCH knockout mice showed the wasting phenotype, indicating that MCH may be involved in obesity (Shimada, M. et al., Nature 396, 670-674, 1998), and identification of MCH receptor have made it possible to search for an antagonist of MCH receptor, which may serve as an anti-obesity agent. Further, it is also reported that urotensin II elicits heart ischemia when administered intravenously to a monkey, and thus it has a strong action on cardiovascular system (Ames, R. S. et al., Nature 401, 282-286, 1999).
As described above, in many cases, orphan receptors and their ligands are involved in novel physiological functions, and thus it is expected that the elucidation thereof may lead to development of a new pharmaceutical. However, because of many difficulties in searching for a ligand of an orphan receptor, even though plenty of orphan receptors have been discovered, only a few thereof are made clear in light of ligand.
Watanabe et al. found a novel receptor SLT as an orphan G-protein-coupled receptor (a protein having the amino acid sequence shown by SEQ ID NO: 3 in the present specification; hereinafter referred to simply as SLT), but it has been unclear until now what is the ligand of the receptor.